In earlier project periods the polyamines, putrescine, spermidine, and spermine, were found to stimulate phosphorylation of four major nonhistone acidic proteins in isolated nuclei from the slime mold Physarum polyycephalum. Two of these phosphoproteins were of nucleolar origin. Their respective Mrs were 70,000 and 52,000. The 70,000 Mr nonhistone acidic protein was purified and many of its putative regulatory properties toward rRNA gene transcription have been identified. During the current award period a polyamine-dependent nonhistone protein kinase which specifically phosphorylates the 70,000 Mr protein will be purified. Optimum conditions will be determined for the preparation of the 70,000 Mr phosphoprotein with this unique protein kinase. The 70,000 Mr phosphoprotein stimulates specific rRNA gene transcription within a transcriptively active minichromosome complex isolable from P. polycephalum nucleoli. The mechanism of the stimulation will be investigated, principally with regard to the possibility of initiating new rRNA chain synthesis. (gamma-32P)ATP and (35S)adenosine 5' -0(3-thiotriphosphate) will be used as probes fmr 5' -end labeling of new rRNA transcripts produced by the isolation minichromosome challenged with exogenously supplied 70,000 Mr phosphoprotein. Additional characterization of the 70,000 Mr phosphoprotein will attempt to determine whether its primary structure contains polyamines covalently bound in amide linkage and whether it is a subunit of the enzyme, ornithine decarboxylase. Past investigations suggest these possibilities. Rabbit antibodies prepare against the 70,000 Mr phosphoprotein will be used to study the cell cycle dependence of phosphorylation of this protein in order to correlate it with temporal scheduling of rRNA synthesis. Additional restriction endonuclease mapping of satellite rDNA will be continued in order to identify nick-translated fragments that contain the binding site(s) for the 70,000 Mr phosphoprotein. Finally, metabolic inhibitors of polyamine biosynthesis and inhibitors of cyclic AMP phosphodiesterase will be used to establish correlations to support the proposal that these natural metabolites control one another's intracellular concentrations.